Acute inflammation is characterized by increased microvascular permeability to plasma proteins and leukocyte recruitment into inflammatory sites. A large increase in permeability of the microvessel wall is a critical event resulting in edema formation and organ dysfunction. The long-term goal of our research is to investigate the mechanisms that regulate microvessel permeability under inflammatory conditions. The objective of this proposal is to investigate the direct correlation between vascular structural changes, transport pathway formations, signal transduction pathways, and the magnitude and time course of the permeability changes in intact microvessels in response to different stimuli. Three specific aims are proposed: 1) Investigate the cellular mechanisms of endothelial gap formation and inflammatory mediator-induced permeability increases in intact microvessels under acute and chronic inflammatory conditions; 2) Investigate the role of pericytes and the basement membranes in the regulation of microvessel permeability under acute and chronic inflammatory conditions; and 3) Identify the cellular mechanisms responsible for ROS-induced permeability increases under acute and chronic inflammatory conditions. These aims will be accomplished using combined confocal microscopy, electron microscopy, with quantitative assessments of microvessel permeability in intact microvessels. Our newly established methods enable us to three- dimensionally visualize and quantify inflammatory mediator-induced gap formation, characterize the changes in endothelial adhesion proteins, as well as to detect changes in actin cytoskeleton in endothelial cells and pericytes in individually perfused microvessels. The electron microscopy study allows ultrastructural changes to be correlated with confocal image findings. The proposed research will provide new information and possibly new concept for a better understanding of the mechanisms that regulate fluid and solute transport when permeability is increased under inflammatory conditions. The insight gained will be directly applied to the identification of an effective target to prevent the permeability increase and contribute to the development of targeted and clinically applicable anti-inflammatory therapies. PUBLIC HEALTH RELEVANCE: The long-term goal of our research is to investigate the cellular mechanisms that regulate permeability in intact microvessels. Accumulated clinical and experimental evidence indicate that an inflammation-associated increase in vascular permeability is the initiating event for a variety of cardiovascular diseases (such as atherosclerosis, diabetes) as well as promoting tumor growth and tumor metastasis. A better understanding of the mechanisms that regulate microvessel permeability is crucial to defining the pathogenesis of many disease conditions, and to aid in the development of novel therapeutic approaches.